Optimizing a biorefinery design for the valorization of Rugulopteryx okamurae by extracting bioactive compounds and enhancing methane production

Research article Optimizing a biorefinery design for the valorization of Rugulopteryx okamurae by extracting bioactive compounds and enhancing methane production David De la Lama-Calvente a,* , María Jos´ e Fern´ andez-Rodríguez b , Rafael Borja a a Spanish National Research Council (CSIC) - Instituto de La Grasa (IG), Department of Food Biotechnology, Campus Universidad Pablo de Olavide, Edificio 46. Ctra. de Utrera, Km 1, 41013 Seville, Spain b Department of Vegetal Biology and Ecology, Faculty of Biology, University of Seville, 41080 Seville, Spain ARTICLE INFO Keywords: Biorefinery Rugulopteryx okamurae Anaerobic digestion Bioactive compounds Antioxidant ABSTRACT The Mediterranean Sea has suffered recently from the unprecedent invasion of the alien macroalga Rugulopteryx okamurae due to global warming and climate change putting at risk the natural local ecosystem. Since 2015 this alga has colonized a great area on the western coast of the Mediterranean Sea and it has been also spotted in other areas such as the Azores Islands or the south of France. The arrival of alga tides into the coasts also provokes collateral environmental problems that need to be addressed. Seaweed-based biorefineries are considered a promising alternative within a circular economy model. This study aims to assess the potential of R. okamurae as raw material for the extraction of reducing sugars (RS) and total polyphenolic compounds (TPC) with antioxidant capacities, the subsequent production of methane from the extracted residue, and the final use of the anaerobic digestate as fertilizer. However, the presence of bioactive compounds greatly varies due to seasonality, location or even natural degradation. In order to provide some insights about these issues, two different batches were assessed: i.e. natural and ashore R. okamurae. As brown algae are characterised by a cell wall composed of crystalline cellulose and lignin, the biomasses studied were mechanically pretreated (dried at 100 ◦C and milled during 60 s) before the single and sequential extraction processes. Results showed that the extraction of the targeted compounds increased by 30–80 % when the biomasses were extracted sequentially. Similarly, the biochemical methane potential of the extracted solid residues increased as the RS and TPC content was reduced (120–150 NL CH4 kg −1 VS ), with no significant impact regarding the biomass origin (natural or ashore) or the extraction process order. An increase in the kinetic constant k (first-order model) of 150% and 75% was observed when the fresh biomass was extracted with water and ethanol, respectively, compared to the value obtained for the unextracted biomass. Finally, the physicochemical characteristics of the different anaerobic digestates generated were assessed for their potential use as biofertilizer. In this study, most of the digestate’ liquid fractions (7 out of 10) comply with European regulations for organic fertilizers and could be used directly. 1. Introduction The increasing recognition of the environmental impact of human activities has compelled a shift from traditional extractive industrial practices towards a more sustainable and eco-friendly approach (Gerdes et al., 2022). Moreover, the environmental repercussions of fossil fuel reliance have spurred international authorities and the scientific community to explore and develop new renewable energy alternatives. Second- and third-generation biomasses, particularly macroalgae, have garnered significant attention over the past decade, and have seen a global production surge to approximately 24 million tonnes in 2022 (Ashokkumar et al., 2022). Macroalgae have emerged as promising resources not only for green energy generation but also for obtaining value-added compounds (Basile and Dalena, 2019). The diverse composition of macroalgae offers a brunch of functional, sensory, and bioactive chemical compounds that hold potential applications across various industries, including food, cosmetics, and pharmaceuticals (Zheng et al., 2022). For instance, several cosmetic formulations use these compounds as additives, excipients, or active ingredients (Zheng et al., 2022). Similarly, polyphenolic compounds are known for their * Corresponding author. E-mail address: [email protected] (D. De la Lama-Calvente). Contents lists available at ScienceDirect Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman https://doi.org/10.1016/j.jenvman.2024.122504 Received 24 January 2024; Received in revised form 8 September 2024; Accepted 12 September 2024 Journal of Environmental Management 370 (2024) 122504 Available online 17 September 2024 0301-4797/© 2024 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). antioxidant activity, with several food applications (Chen et al., 2023), and, additionally, they play a fundamental role in the humification process of biomasses (Zhang et al., 2023). The utilization of these compounds derived from seaweed in industrial processes underlines their extensive study and documentation in the scientific literature. Nevertheless, these industrial applications are mostly based on cultivated seaweed (Zheng et al., 2022) which presents several drawbacks, e.g.: it entails the building of specific facilities when cultivated in-land; it may contribute to eutrophication when cultivated off-shore within the marine ecosystem; etc. (Khan et al., 2023). Thus, a more sustainable approach would be to take advantage of naturally produced macroalgae biomass, although these systems would be associated with other challenges as well (e.g. collection costs and logistics, inconsistent production, compositional variation, etc). In the last years, the spread of invasive seaweed species and the frequency of the so-called green tides have increased worldwide due to climate change and coastal areas eutrophication (Ravaglioli et al., 2022; Xia et al., 2022). These phenomena are considered ones of the main threats to marine ecosystems’ functioning, native wildlife, economics, and human health (Pereira et al., 2021; Salimi et al., 2021). Among these macroalgae species, two seaweed have focused the attention due to the extension of their impact: the Sargassum invasion in the Caribbean (Lopresto et al., 2022); the Rugulopteryx okamurae (R.o.) invasion in the Mediterranean Sea (Roca et al., 2022). The brown alga R.o. has colonized a great part of the Mediterranean coasts in Spain and Morocco since 2015, when it was first detected in Ceuta (El Aamri et al., 2018; García-G´ omez et al., 2020). Its rapid and uncontrolled spread has provoked a severe ecological impact associated with unprecedented biodiversity loss (García-G´ omez et al., 2020). Besides, the loosed biomass ends up on the shores, where is usually left for months, causing environmental pollution and costing millions to local authorities due to waste management and the loss in the fishery sector (B´ aez et al., 2023; MITECO, 2022). A sustainable and economically feasible solution is needed in order to palliate the environmental effect of this plague and its impact on anthropogenic activities. The studies on this invasive species has increased in the last years. Some studies, prior to its appearance in Ceuta, already highlighted the presence of secondary metabolites, such as terpenes, and the high α -glucosidase inhibitory activity of its ethanolic extracts (Jeong et al., 2012; Suzuki et al., 2002). More recently, it has been discovered that R. okumare produces diterpenoids with a high anti-inflammatory activity. One of them, a chemical weapon namely dilkamural, was presented as harmful and even lethal to sea urchins (Casal-Porras et al., 2021; Cuevas et al., 2021). However, in terms of bioactive compounds extraction methodologies, the reported studies have focused on the production and extraction of carbohydrates and antioxidant compounds. For instance, the alga has been fermented with the fungus Aspergillus awamori followed by enzymatic hydrolysis allowing the production of 240 g reducing sugars kg −1 dw (Agabo-García et al., 2023). In a different study using only commercial enzymes the highest yield reported was 13.7 g sugars L −1 in the hydrolysates (Romero-Vargas et al., 2023). From a different perspective, the relevance of drying related to the extraction of alginate and antioxidant compounds has also been assessed by comparing the effect of sun drying, conventional oven and microwave hydrodiffusion and gravity (MHG) (L´ opez-Hortas et al., 2023). Furthermore, the dried samples were extracted with an ethanol: water solution (1:1 (v/v)) for 24 h at room temperature and 200 rpm. Results showed that MHG was the best alternative providing the highest antioxidant and alginate fraction (L´ opez-Hortas et al., 2023). Earlier experiments carried out by Kurata et al. (1988) showed that the use of alcoholic solvents, such as methanol, was able to extract spatane-type diterpenes with feeding-deterrent activity from the alga R. okamurae. Later, the ester ethyl acetate was used to recover a newly isolated secospastane namely dilkamural, and acetone was used for two novel diterpenoids with feeding-deterrent activity (Ninomiya et al., 1999; Suzuki et al., 2002). More recently, the ethanolic extract of the R. okamurae was tested for the α -glucosidase inhibitory activity, showing an inhibition of 90.9 ±1.02 % and placing this alga at the fourth highest position when compared with other 19 species (Jeong et al., 2012). Casal-Porras et al. (2021) used an acetone/methanol (1:1 v/v) mixture followed by diethyl ether to extract secondary metabolites (such as Dilkamural) and tested their feeding-deterrent activity. Using the same extraction protocol, more than ten new diterpenoids were isolated, among which some of them showed strong anti-inflammatory behaviour (Cuevas et al., 2021, 2023). Furthermore, it has been reported that while the ethanolic (H 2 O:EtOH 1:4) and the methanolic (H 2 O:MeOH 1:4) extracts of R.o. presented some inhibition to some human pathogens (Staphylococcus aureus and Cutibacterium acnes), with no significant differences between them, this effect was not observed with fish pathogens (Vega et al., 2023). Moreover, the aqueous extract showed no inhibition to either human or fish pathogens (Vega et al., 2023). However, the aqueous extract obtained by microwave-assisted hydrothermal (MAH) processing showed a 53–65 % (depending on the temperature applied) inhibitory efficacy to cell growth in tumoral cell line HeLa 229 (Ferreira-Anta et al., 2023). Additionally, this MAH method allowed for the extraction of alginate (3.2 % recovery) and phenolic compounds (Ferreira-Anta et al., 2023). Similarly, the microwave hydrodiffusion and gravity treatment enable the recovery of a gelling fraction and antioxidants (L´ opez-Hortas et al., 2023). All these evidences proved the potential of R. okamurae to provide for bio-active compounds. However, the use of chemicals (e.g. MeOH or diethyl ether) or temperature during the extraction process may discourage scaling up to a biorefinery industry due to the high costs involved. Despite the use of the invasive species R. okamurae as a feedstock for the obtention of added-value compounds with bioactive properties, other studies have highlighted its potential as raw material for the production of compost, bioplastics or aquafeeds for European sea bass (Fonseca et al., 2023; Pat´ on et al., 2023; Santana et al., 2022). Furthermore, the potential as feedstock for energy production has also been assessed. Hydrogen and short-chain fatty acids production through dark fermentation after microwave pre-treatment has been considered a promising alternative (Fern´ andez-Medina et al., 2022). Hydrogen is a great alternative to fossil energy sources because it has a very high energy content and is a clean and green energy source. In addition, it shows high energy efficiency and compatibility with fuel cells as well as its renewal waste product, i.e. water (Zinatloo-Ajabshir et al., 2020a, 2020b, 2023). In this sense, it has been recently reported that lanthanum zirconate ceramic microstructure exhibits an improved electrochemical hydrogen storage performance compared to the Nd 2 Zr 2 O structure due to its higher specific surface, as well as its more favorable physical and chemical nature (Zinatloo-Ajabshir et al., 2023). Other research carried out by this same research group also demonstrated the suitability of the green Nd 2 Sn 2 O 7 nanostructures, synthesized using palm extract, for electrochemical hydrogen storage (Zinatloo-Ajabshir et al., 2020a). Moreover, Zinatloo-Ajabshir et al. have also shown the benefits of the use of dysprosium stannate nanoparticles using Ficus carica extract as photocatalyst for the degradation of organic pollutants (i.e. acid violet 7 and crystal violet) from water under visible irradiation (Zinatloo-Ajabshir et al., 2020b). However, the production of biomethane from this invasive species has been more deeply assessed. For instance, the use of R. okamurae as co-substrate with olive mill solid waste (OMSW) in a 1:3 mixture (VS basis) produced 26.8 % more methane than the mono-digestion of both substrates (De la Lama-Calvente et al., 2023a); moreover, a preliminary economic assessment showed that the zeolite-assisted milling pre-treatment on R. okumare could potentially increases the income of a full-industrial plant, treating only algal biomass, by a 43 % (De la Lama-Calvente et al., 2023a). This article attempts, for the first time, to propose a comprehensive management system for the disposal and valorization of an invasive macroalga (Rugulopteryx okamurae) within a closed-loop circular economy approach. Firstly, the extraction efficiency of value-added D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 2 compounds (i.e. reducing sugars (RS) and total polyphenolic compounds (TPC)) by different solvents (i.e. water and ethanol at room temperature), and the antioxidant capacity of the obtained extracts were evaluated. Furthermore, the biomethane potential of the non-extracted alga and the extracted solid residues have been performed by biochemical methane potential (BMP) tests, while comparing simultaneously two algae batches collected at different stages of their life cycle (i.e. from their natural location linked to sea bottom rocks and recently arrived at the coast after loosening from its natural location). The effect on the kinetic parameters has been also discussed. Finally, the anaerobic digestates have been analyzed and compared with local regulations to discuss their potential use as fertilizer or soil amender. 2. Materials and methods 2.1. Seaweed The marine algal biomass of R.o. was provided by the Laboratory of Marine Biology of the University of Seville from two different spots along the Algeciras coast (36◦03 ′ 0’’N 5◦29 ′ 7’’W - 36◦03 ′ 7’’N 5◦14 ′ 8’’W): i) Fresh R.o. (FR) attached to the sea bottom rocks in its natural environment; ii) Fresh Ashore R.o. (FAR), immediately collected once the loosen biomass reaches the beach. Samples were washed in situ with seawater to eliminate debris and stored at −20 ◦C upon their arrival at the laboratories. Prior to further use, samples were thawed at 4 ◦C for 2 h and washed again with deionized water in order to eliminate the interference of marine salts and other potential contaminants during the experiments. Details of the washing effect on the elemental content are provided in Table S1 of the supplementary material. 2.2. Value-added compounds extraction process Based on a preliminary study (De la Lama-Calvente et al., 2024) the optimum extraction process was set as follows: the algal biomass should be oven-dried at 100 ◦C and milled during 60 s. For the single extraction process, 0.5 g VS of the dried and milled biomass was mixed with 25 ml of solvent (water or ethanol) and stirred (250 rpm) during 1 h at room temperature (25 ◦C aprox.). Then, the mixture was filtrated (Whatman, 0.45 μ m pore size nylon filter), and the liquid fraction was immediately storage at −20 ◦C until further use while the solid residue was further oven-dried at 100 ◦C for a period of 24 h. The same protocol was implemented in the second extraction step for the sequentially extracted tests as it is shown in Fig. 1. Briefly, preliminary studies based on response-surface methodology indicated that the selected sample conditioning enhances the extraction process. This was mainly due to the major cell wall disruption occurring in comparison with less severe treatments. Middle temperatures (100–120 ◦C) proved to reduce significantly the cellulose crystallinity of the alga cell wall, which favoured the cellulose breakdown during the milling process (De la Lama-Calvente et al., 2023a). The selection of the two solvents was based on preliminary studies and literature review: Water is commonly the most economical solvent, however, it has limited affinity for some interesting compounds, such as phenols, terpenes, lipids, etc. On the other hand, ethanol, although more expensive is also widely used as it could be easily reusable, additionally, it can extract a wider range of compounds, such as pigments, polyphenols, etc. 2.3. Anaerobic inoculum biomass The anaerobic inoculum was collected from a brewery wastewater anaerobic treatment plant located in Seville (Heineken) and the same batch was used for all the biomethanization assays. Immediately after the reception of this inoculum at the laboratory facilities, the sludge was placed in a water bath at 37 ±2 ◦C for 36 h in order to lessen the endogenous methane production. The correct anaerobic activity of the inoculum was tested by placing positive controls (cellulose; Avicel® PH- 101, Fluka) during the assays and confirming that at least 80 % of the theoretical methane yield was achieved (Holliger et al., 2016). The main physicochemical parameters of the anaerobic inoculum were as follows: TS: 54.8 ±0.4 g kg −1 ; VS: 42.0 ±0.4 g kg −1 ; MS: 12.7 ±0.2; tCOD: 42 ±4 g O 2 kg −1 ; pH: 7.59 ±0.05; TA: 3600 ±200 mg CaCO 3 L −1 . 2.4. Biochemical methane potential test The BMP test was carried out by following the indications described by Holliger et al. (2016). Briefly, the assay was performed at mesophilic temperature (37 ±2 ◦C) and at an inoculum-to-substrate ratio (ISR) of 2 (VS) reaching a final concentration of 24 g VS L −1 in the reactors. As established in Holliger et al. (2016), the recommended ISR values are between 2 and 4, where higher values are recommended for Fig. 1. Schematic design of the biorefinery conceptualization carried out. Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly with ethanol and then with water (FR-EW). BMP: Biochemical methane potential; RS: Reducing Sugars; TPC: Total Phenolic Content; FRAP: Ferric Reducing Antioxidant Power; DPPH RSA : 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Activity. D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 3 easy-to-degrade substances and lower values for substrates with recalcitrant components (such as cellulose or lignin). Thus, the ISR was established based on the potential presence of hard-to-degrade components in the selected biomass. The 250 mL working volume consisted of the inoculum, the substrate, and distilled water. The reactor’s headspace was maintained at approximately 10 % of the reactor’s total volume. Each test, including blanks and positive controls, was carried out in triplicate, and steadily stirred at 300–400 rpm. Prior to the start-up of the experiment, nitrogen gas was flushed into each reactor to guarantee an anoxic environment. Finally, reactors were placed in a water bath where the temperature was maintained at 37 ±2 ◦C by using a thermostat (Termotronic, Selecta, Spain). The tests were considered finished when the daily measured methane was <2 % of the accumulated value for three days in a row. This period was c.a. 28 days. Methane was measured volumetrically after passing the produced biogas through a 2 N NaOH solution to capture the generated CO 2 . The accumulated biomethane yields were then calculated by subtracting the endogenous methane production from the blanks and by normalizing the volume to standard temperature and pressure conditions (273.15 K; 101.325 kPa). In total, 10 different tests were carried out (Fig. 1): (FR) Fresh R. okamurae; (FR-W) Fresh R. okamurae extracted with water; (FR-E) Fresh R. okamurae extracted with ethanol; (FR-WE) Fresh R. okamurae extracted with water and then with ethanol; (FR-EW) Fresh R. okamurae extracted with ethanol and then with water; (FAR) Fresh ashore R. okamurae; (FAR-W) Fresh ashore R. okamurae extracted with water; (FAR-E) Fresh ashore R. okamurae extracted with ethanol; (FAR-WE) Fresh ashore R. okamurae extracted with water and then with ethanol; (FAR-EW): Fresh ashore R. okamurae extracted with ethanol and then with water. 2.5. Analytical methods The main physicochemical characteristics of the algal biomasses before and after the extraction processes and the anaerobic sludge were analyzed prior to the biomethanization tests (Table 1). The anaerobic digestates resulted from the biochemical methane potential (BMP) tests (Table 2) and the liquid fractions obtained after the extraction processes (Table 3) were analyzed as well. For the analysis of soluble parameters, samples were previously centrifugated (12,000 rpm, 10 min) and filtrated (Whatman, 0.45 μ m pore size nylon fiber filter). All the analyses were carried out at least in triplicates. The standard methods 2540B & 2540E were followed in order to obtain the total solids (TS), volatile solids (VS), and mineral solids (MS) fractions (APHA, 2017). Chemical oxygen demand (COD) was determined by following the indications in Raposo et al. (2008) and the standard method 5220D (APHA, 2017), for total (tCOD) and soluble (sCOD) fractions, respectively. pH and total alkalinity (TA) were determined by using a pH meter model Crison and by titration to pH 4.3 as shown in the 2320B standard method for TA (APHA, 2017). Total ammonia nitrogen (TAN) was performed by distillation and titration following the standard method 4500-NH 3 (APHA, 2017). Metal elements were determined by digesting the samples with a Mars Xtraction microwave (CEM, USA) and analyzed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS, AGILENT 7800, Spain) as described in the U.S. EPA 3051A method (USEPA, 2007). The following methods were carried out spectrophotometrically in a 96-well microplate using a microplate multiscan reader (Bio-RAD, iMark™). Total phenolic content (TPC) was determined by carrying out the Folin-Ciocalteau method using gallic acid (Sigma-Aldrich) as standard and measuring the absorbance at 750 nm (Magalh˜ aes et al., 2010; Zhang et al., 2006). Reducing sugars content (RS) was determined by the Anthrone method using glucose (PanReac) as standard and measuring the absorbance at 630 nm (Leyva et al., 2008). The DPPH (stable radical of 2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity test (DPPH RAS ) was performed by calculating the efficient concentration (EC 50 ) of the extract expressed as the amount of antioxidant per mL able to reduce the DPPH (0.2 mM) (Sigma-Aldrich) initial absorbance at 490 nm by 50% (Rodríguez et al., 2005). The ferric reducing antioxidant power assay (FRAP) was carried out using Trolox (6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid) (Sigma, ≥97 %) as standard and measuring the absorbance at 490 nm; results are given as Trolox mM equivalents (Benzie and Strain, 1996). 2.6. Data analysis 2.6.1. Biodegradability The biodegradability of each substrate was calculated by the CODt analysis as proposed by Nielfa et al. (2015). The theoretical methane yields and the biodegradability results after each BMP test are displayed in Table 2. 2.6.2. Kinetic models 2.6.2.1. First-order kinetic model. The kinetic behaviour and the process performance of the AD of studied algal biomasses (fresh and ashore) were analyzed by the following first-order kinetic model: G =G max ⋅[1 – exp (-k⋅t)] (1) Where G is the specific cumulative methane production (L CH4 kg −1 VS ), G max is the ultimate methane production (L CH4 kg −1 VS ), k is the specific rate constant (days −1 ) and t is the digestion time (days). This kinetic model is normally applied to assess the kinetics of the batch AD processes for different types of biodegradable substrates (Li et al., 2012; Scarcelli et al., 2020). This model assumes that the microbial cell mass does not affect the methane production, which is treated as only-proportional to the amount of substrate (Wang et al., 2017). 2.6.2.2. Modified Gompertz model. The Modified Gompertz model was assessed as suitable to fit the experimental data recorded by the BMP tests: B =B max *exp[-exp(R max *exp(1)*(ℷ - t))/B max +1)] (2) Where B (L CH4 kg −1 VS ) is the cumulative specific methane production, B max (L CH4 kg −1 VS ) is the ultimate methane production, R max is the maximum methane production rate (L CH4 (kg VS ⋅d) −1 ), t (d) is the digestion time and ℷ is the lag time (d). This model assumes that Table 1 Main physicochemical characteristics of the substrates used for the biochemical methane potential tests. Values represent mean ±standard deviation. Substrates VS MS TAN tCOD (% TS) (% TS) (mg N-NH 3 kg −1 ) (g O 2 kg −1 ) FAR 89 ±1 a,b 11 ±1 a,b 1140 ±40 a 1160 ±20 a,b FAR-W 90.2 ±0.9 b 9.8 ±0.9 a 1250 ±70 a,b 1160 ±50 a,b FAR-E 88.4 ±0.5 a,b 11.6 ±0.5 a,b 1400 ±200 a,b 1190 ±30 a,b FAR-WE 89.4 ±0.7 a,b 10.6 ±0.7 a,b 1200 ±100 a,b 1150 ±30 a FAR-EW 89.9 ±0.5 a,b 10.1 ±0.5 a,b 1420 ±80 a,b 1140 ±30 a FR 89 ±1 a,b 11 ±1 a,b 1100 ±100 a 1270 ±10 b FR-W 91.05 ±0.05 b 8.95 ±0.05 a 1130 ±50 a 1336 ±3 c FR-E 87 ±3 a 13 ±3 b 1340 ±30 a,b 1180 ±10 a,b FR-WE 90.8 ±0.6 b 9.2 ±0.6 a 1400 ±100 a,b 1180 ±30 a,b FR-EW 89.7 ±0.6 a,b 10.3 ±0.6 a,b 1500 ±200 b 1183 ±9 a,b Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly with ethanol and then with water (FR-EW). VS: Volatile Solids; MS: Minerals Solids; TAN: Total Ammonium Nitrogen; tCOD: Total Chemical Oxygen Demand. Different superscripted letters in the same column mean values are significantly different. D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 4 methane produced in the batch modes is directly related to the specific methanogenic bacterial growth (Donoso-Bravo et al., 2010; Li et al., 2012). The model was thus used to predict the trend in microbial growth and simulate the cumulative methane yield. The goodness-of-fit and the accuracy of the results for both models have been assessed by the determination coefficients (R 2 ) and the standard errors of estimates (S.E. E.). The kinetic parameters for each experiment and mathematical adjustment were determined numerically from the experimental data obtained by non-linear regression using the software Sigma-Plot (v. 11). 2.6.3. Statistical analysis Analytical tests and BMP tests were at least carried out in triplicates and values are given by means ±standard deviations. Where corresponded, a two-tale Student’s t-test and the one-way analysis of variance (ANOVA) were carried out. Additionally, the Tukey’s test was performed as a post-hoc analysis. For the purposes of data discussion, a confidence level of α =0.05 was accepted as not statistically significant. All the statistical analyses were carried out by SPSS Statistics software (v29, IBM, Business Machines Corp.). Table 2 Main physicochemical characteristics of the digestates derived from the BMP test. Experimental and theoretical biomethane yields and the biodegradability achieved. Values represent mean ±standard deviation. Substrates pH TA TAN TANs TS VS MS tCOD sCOD BMP exp BMP th BD CODt (mg CaCO 3 kg −1 ) (mg N-NH 3 kg −1 ) (mg N- NH 3 L −1 ) (g kg −1 ) (% TS) (% TS) (g O 2 kg −1 ) (mg O 2 L −1 ) (NL CH4 kg −1 VS ) CODt (L CH4 kg −1 VS ) (%) FAR 7.70 ± 0.02 a,b 3540 ±20 a 640 ±20 a,b 490 ±20 a 23.7 ± 0.3 a 77.4 ± 0.7 a 22.6 ± 7 a 29.6 ± 0.1 a 1550 ± 80 a 100 ±6 a,b 454 22 % FAR-W 7.73 ± 0.00 b 3840 ±70 b, c 682 ±2 b 560 ±9 b 24.2 ± 0.6 a 79 ±3 a,b 21 ±3 a 29.3 ± 0.9 a,b 2300 ± 200 b 100 ±20 a, b,c 452 22 % FAR-E 7.78 ± 0.01 c 3940 ±80 c 690 ±10 b 560 ±10 b 24.9 ± 0.4 a 78 ±1 b 22 ±1 a 23 ±1 c 2910 ± 80 c 110 ±10 a, b,c,d 473 23 % FAR-WE 7.627 ± 0.006 d 3960 ±30 b 682 ±4 b 550 ±10 a, b 24.9 ± 0.1 a 78.2 ± 0.7 b 21.8 ± 0.7 a 28.3 ± 0.3 a,b,d 2300 ± 100 b 150 ±20 c, d 451 33 % FAR-EW 7.79 ± 0.00 c 4250 ±90 d 760 ±20 c 650 ±30 c 24.7 ± 0.2 a 77.9 ± 0.6 a,b 22.1 ± 0.6 a 27.7 ± 0.3 b,d 2400 ± 100 b 150 ±20 d 443 34 % FR 7.68 ± 0.01 a 3550 ±40 a 630 ±30 a,b 513 ±9 a,b 25 ±1 a 78 ±5 a,b 22 ±5 a 29.5 ± 0.5 a,b 2800 ± 30 c 90 ±20 a 499 18 % FR-W 7.70 ± 0.00 a 3650 ±60 a, b 590 ±40 a 495 ±9 a 24.2 ± 0.4 a 78 ±1 a,b 22 ±1 a 30.0 ± 0.6 a 2370 ± 80 b 115 ±5 a,b, c,d 515 22 % FR-E 7.79 ± 0.01 c 3800 ±90 b, c 670 ±20 b 530 ±40 a, b 24.5 ± 0.6 a 80 ±1 b 20 ±1 a 28.8 ± 0.4 a,b,d 2300 ± 100 b 121 ±3 a,b, c,d 479 25 % FR-WE 7.81 ± 0.02 c 3700 ± 100 a,b 630 ±30 a,b 513 ±9 a,b 24.8 ± 0.5 a 78 ±1 b 22 ±1 a 29.0 ± 0.3 a,b,d 2100 ± 100 b 140 ±10 b,c,d 454 31 % FR-EW 7.90 ± 0.01 e 3780 ±50 b, c 660 ±10 a,b 520 ±30 a, b 24.5 ± 0.4 a 79 ±1 a,b 21 ±1 a 27.5 ± 0.4 d 2400 ± 200 b 140 ±10 b,c,d 463 30 % Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly with ethanol and then with water (FR-EW). TA: Total Alkalinity; TANt: Total Ammonium Nitrogen of the whole digestate; TANs: Total Ammonium Nitrogen of the liquid fraction of the digestate; TS: Total Solids; VS: Volatile Solids; MS: Minerals Solids; tCOD: Total Chemical Oxygen Demand; sCOD: Soluble Chemical Oxygen Demand. Different superscripted letters in the same column mean values are significantly different. Table 3 Main physicochemical characteristics of the liquid extracts. Extracted added-value compounds concentration and antioxidant properties. Values represent mean ± standard deviation. FAR-W FAR-E FAR-WE a FAR-EW a FR-W FR-E FR-WE a FR-EW a VS (% TS) 95 ±8 a,b 95 ±9 a,c 100 ±0 c 70 ±20 a,b,c 80 ±20 a,b,c 81 ±8 a,b 99 ±2 a,b,c 90 ±10 b pH 7.22 ±0.02 a 7.11 ±0.05 a 7.49 ±0.08 b 7.57 ±0.06 b 5.67 ±0.04 c 5.44 ±0.03 d 5.90 ±0.06 e 5.893 ±0.006 e TA (g CaCO3 kg ¡1 )260 ±20 a 88 ±9 b 75 ±1 b 240 ±20 a 240 ±10 a 75 ±4 b 70 ±4 b 203 ±9 b sCOD (mg O 2 L ¡1 )1849 ±30 a,b 1700 ±100 a 760 ±60 c 2100 ±100 b 5700 ±200 d 4500 ±200 e 2800 ±100 f 6290 ±40 g TAN (mg N-NH 3 L ¡1 )n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. RS (g Glucose kg VS ¡1 )11.6 ±0.6 a,b 2.3 ±0.3 c 0.7 ±0.1 c 16 ±2 d 13.3 ±0.9 b,d 8.0 ±0.7 a,e 4.7 ±0.6 e 32 ±5 f TPC (g Galic Acid kg VS ¡1 )2.90 ±0.09 a 13.3 ±0.3 b 20.8 ±0.9 c 3.34 ±0.05 a 2.04 ±0.03 a 22 ±2 c 21 ±2 c 2.95 ±0.08 a FRAP (mM Trolox eq.) 1.50 ±0.04 a 0.76 ±0.05 b 0.863 ± 0.009 c 1.56 ±0.01 a 1.05 ±0.04 d 0.89 ±0.02 c 0.91 ±0.01 c 1.16 ±0.04 e DPPH RSA (IC 50 – mg TS ml ¡1 ) 0.144 ±0.007 a, b 0.15 ±0.01 a 0.12 ±0.02 a,b, c 0.098 ± 0.004 c 0.118 ±0.004 b, c 0.29 ±0.01 d 0.27 ±0.04 d 0.132 ±0.006 a, b Fresh ashore Rugulopteryx okamurae extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly with ethanol and then with water (FR-EW). VS: Volatile Solids; TS: Total Solids; TA: Total Alkalinity; sCOD: Soluble Chemical Oxygen Demand; TAN: Total Ammonium Nitrogen; RS: Reducing Sugars; TPC: Total Phenolic Content; FRAP: Ferric Reducing Antioxidant Power; DPPH RSA : 2,2-Diphenyl-1-picrylhydrazyl Radical Scavenging Activity. Different superscripted letters in the same row mean values are significantly different. n.d.: not detected. a Results are related to the liquid fraction of the second extraction process. D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 5 3. Results and discussion 3.1. Rugulopteryx okamurae extraction yields 3.1.1. Physicochemical characteristics of the Rugulopteryx okamurae extracts Table 3 shows the physicochemical characteristics of the different extracts assessed after the extraction optimization process. The extracted organic fraction in terms of sCOD showed significant differences among samples. In fact, the extracts from the ashored alga (FAR) presented lower values (from 760 ±60 to 2100 ±100 mg O 2 L −1 ) than those from the natural equivalent (FR) (from 2800 ±100 to 6290 ±40 mg O 2 L −1 ). These results are in accordance with the RS content of the extracts, which were higher in the FR samples than in their FAR equivalents. In addition, other compounds not tested, such as pigments, are expected to be present in FR samples at higher concentrations, as these compounds are highly sensible to degradation due to light incidence or temperature (Cikoˇ s et al., 2022). Finally, the organic matter detected by the sCOD analysis showed a higher affinity to water than to ethanol. However, when the accumulated values were compared, it seems that the highest value was obtained when ethanol was used before water, regardless the sample was the FAR or the FR. In fact, the sum of FAR-E and FAR-EW sCOD values was a 46 % higher than the sum of FAR-W and FAR-WE. Similarly, the sum of FR-E and FR-EW sCOD values was a 27 % higher than the sum of FR-W and FR-WE, which, support the idea of ethanol as a significant cell disruptor. The different elements founded in the mineral fraction are detailed in Table S2 of the supplementary material. Although the content of most of the economically interesting metals (i.e. Cu, Cd, Cr, Ti) were present at low concentrations (≈ppb), their recovery may be a possible path worth to investigate. Unfortunately, studies assessing the valorization of macroalgae through metal recovery are scarce. However, the metal recovery from other wastewaters or contaminated soils has been widely studied. For instance, a stable electrocatalyst-coupled with super-stable mineralizer structure showed a recovery yield of 91.1 % for Cu 2+ (Liu et al., 2024). Other techniques, such as flow-electrode capacitive deionization, showed less selectivity and produced low-quality metal products. However, if coupled with a liquid membrane chamber, then the outcome improved significantly (Wu et al., 2024). In any case, the use of adsorbents is the most spread technique in the removal of heavy metal from wastewaters (Rakib et al., 2021). These results suggest that the valorization of macroalgae through the extraction of heavy metals could be a promising pathway which need further investigation. Regarding the acidity of the extracts, significant differences were observed between the two samples assessed. While the FAR samples showed neutral pH values (from 7.11 ±0.05 to 7.57 ±0.06), the FR samples showed an acid pH (from 5.44 ±0.03 to 5.90 ±0.06). Lesser acute differences were observed between one-step extracts and two-step extracts, although they were statistically significant with the pH slightly increasing, for both FAR and FR, after the second extraction. Similarly, ethanolic extracts showed slightly lower pH values than the aqueous equivalents across the board. These findings were also supported by the TA values of the extracts, although, no great differences were observed between the two types of algal biomass studied. This acidity difference observed between the fresh and the ashore biomass could also explain the different TPC and RS yields obtained. In fact, the use of diluted acids improves the oligosaccharides solubilization (Andrade et al., 2022), while, by contrast, tend to increase the protein precipitation (Veide Vilg and Undeland, 2017). However, these results are in contrast with previous studies suggesting that the composting process of seaweed tends to reduce the pH value (Walsh and Waliczek, 2020). Reasons behind this may be due to the chemical composition of R. okamurae and the presence of specific acidic secondary metabolites (e.g. diterpenes) which are eliminated during the natural degradation process. This would also explain the lower pH of ethanolic extracts as secondary metabolites, such as terpenes, are usually not-water soluble (Jiang et al., 2017). Finally, all the tested liquid samples showed no TAN content. This suggests that the extraction process was not able to extract protein-derived compounds as these are usually soluble in aqueous solution at alkaline pH’s (Veide Vilg and Undeland, 2017). 3.1.2. Total phenolic compound yields after single extraction The higher TPC values were obtained when ethanol was used as solvent in contrast with those values obtained from the aqueous liquid extracts (Table 3). In fact, using water as a solvent would reduce the TPC yield by a 78–90 %, depending on the biomass. These results were in agreement with other studies showing that alcoholic solvents (such as ethanol or methanol) present higher affinity to phenolic compounds than aqueous solutions (Vega et al., 2023). The phenolic compounds values obtained in the present study (2.04 ±0.03–22 ±2 g gallic acid kg −1 VS ) are in the same range of other brown seaweeds (0.2–235 g gallic acid kg dw ) (Barciela et al., 2023; Getachew et al., 2020) and very similar to those previously reported for R.o. using a mixture of H 2 O:EtOH (1:1) as solvent (≈20 g kg −1 dw ) (Vega et al., 2023). However, when the alga R.o. was subjected to a hydrothermal pretreatment by microwave irradiation (220 ◦C during 20 min) the maximum TPC yield (0.034 g gallic acid kg −1 VS ) was significantly lower than the reported in the present study (Fern´ andez-Medina et al., 2022). While there were no significant differences between the aqueous extract of the two R.o. samples, the ethanolic extracts differed greatly, being the FR-E sample which showed the highest amount. This sample represents healthy individuals during the summer period, which have been pointed as the peak season for the content of phlorotannins in brown algae, concurring with the reproductive season (Connan et al., 2004; Imbs and Zvyagintseva, 2018). However, the TPC also depends on the habitat of the algae, the intensity of illumination, or, as shown in this study, on the natural degradation, among other factors (Imbs and Zvyagintseva, 2018). Although, the reduction of TPC during the natural degradation of marine algae have not been yet assessed, studies on the composting of similar biomasses could provide approximated information. For example, after the vermicomposting process of oil palm empty fruit bunches, the phenolic compounds decreased from 311 g galic acid kg −1 to 15 g galic acid kg −1 , meaning more than a 95 % elimination of phenolic compounds (Yahaya et al., 2017). Moreover, during the senescence of the cells of the brown alga Ectocarpus sp. all the cell organelles start to degrade as the cell ages up to the cell death when some of the organelles, like the nucleus or the Golgi apparatus, are completely banished (Oliveira and Bisalputra, 1977). In this study, naturally degraded FAR samples presented only a 40 % less TPC than FR. This could also indicate the freshness of the FAR samples, which were taken immediately once reached the coasts. And suggests that the TPC of the completely sun-dried algal biomass found in the coasts could be negligible. As a result, extraction with pure water aiming for phenolic compounds, except for economic reasons, seems to not be adequate and, furthermore, the algae should be as fresh as possible. 3.1.3. Reducing sugars yields after single extraction Opposite to TPC results, water was a better solvent than alcohol for the RS extraction (Table 3). In fact, the use of ethanol would deplete the RS yield by a 40–80 %, depending on the biomass. Similar results have been widely reported in the literature stating that RS present higher affinity to aqueous solutions. For example, when the terrestrial plant Vernonia amygdalina was extracted with methanol, ethanol and aqueous solutions, sugars were only found in the aqueous fraction (Hussen and Endalew, 2023). Similarly, the RS extraction of the blue-green microalgae Oscillatoria limosa was more efficient with water than with methanol or ethanol, however, acetone showed the highest affinity (Tunio et al., 2022). This is the first time that the RS content of R.o. has been reported after a sole aqueous or ethanolic extraction process without the application of thermal energy. Nevertheless, when the alga R.o. was subjected to a hydrothermal pretreatment by microwave irradiation (200 ◦C D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 6 during 15 min) the maximum RS yield (0.060 g glucose kg −1 VS ) (Fern´ andez-Medina et al., 2022) was significantly lower than the reported in the present study (from 0.7 ±0.1 to 32 ±5 g glucose kg −1 VS ). Other studies have assessed the potential of R.o. as a source of monosaccharides by enzymatic saccharification. In this sense, Agabo-García et al. (2023) reported values of 1.5–4.27 g L −1 hydrosilate after a biological treatment (5–12 days) followed by enzymatic hydrolysis (24 h). Moreover, values of 12.22 g L −1 hydrosilate have been reported after the hydrolytically pretreated biomass by CellicCTec2 containing cellulase and endo-β-1,4-xylanase (Romero-Vargas et al., 2023). Similar results (12 g L −1 hydrosilate ) were obtained by Fern´ andez-Medina et al. (2022) when the enzymatic pretreatment with CellicCTec2 was applied after a hydrothermal pretreatment by microwave irradiation. In the present study, without a saccharification process of any kind, the concentration of reducing sugars in the liquid fraction was significantly lower, ranging from 14 to 640 mg L −1 extracted liquid . This confirms the great potential of the mentioned pretreatments to enhance the conversion of carbohydrates into shorter-chain oligosaccharides. Nevertheless, this study aimed to assess the potential of the alga R.o. as a feasible feedstock within a biorefinery industry based on eco-friendly and low-cost processes, which, as expected, impairs the added-value compound extraction yield. However, the energetic valorization of the extracted residue may overcome any loss during the extraction process. Regarding the biomass type, FR samples showed a higher RS content in both ethanolic and aqueous extracts. Significantly, the ethanolic extract presented an RS content 2.5 times higher than that found in the FAR samples, while for the aqueous extract, the increment represented only 15 %. Similar to the TPC, the carbohydrate content of algae peaks during late spring to middle summer, although its proportion may be altered by several factors, such as: i) temperature; ii) light irradiance; iii) nutrient availability; iv) seawater hydrodynamics; v) day length (Schiener et al., 2014). To the best of our knowledge, the effect of natural degradation on the RS content of marine algae has not been reported earlier. However, several studies on the composting of macroalgae confirmed that this process reduces and breakdown the organic matter content, being the short-chain carbohydrates the easier biodegradable fraction (Oliveira and Bisalputra, 1977; Pat´ on et al., 2023; Walsh and Waliczek, 2020). 3.1.4. Combined TPC and RS yields in sequential extraction processes It may be clear that the best methodology should be able to provide with the highest recovery yield for both TPC and RS in a one step extraction process. However, while ethanol is presented as the best option for the recovery of phenolic compounds, water would be selected to obtain the highest RS yield. These results suggested that a single step extraction process should be focused on the extraction of one type of compounds, and that a cascade system could be a more recommended option for an efficient extraction of both TPC and RS. Thus, a sequential two-step extraction process would be performed in order to simulate a more comprehensive biorefinery system where the biomass is exhausted from most of its value-added compounds. The sequential extractions with water or ethanol have been evaluated and the results are shown in Table 3. Regarding the TPC, no significant differences were observed for the FR samples between the first extracted fraction and the second extracted fraction, regardless of the solvent. For example, the one-step aqueous extract (FR-W) resulted in a TPC of 2.04 ±0.03 g glucose kg −1 VS , while the second aqueous extract (after the ethanolic extraction) (FR-EW) resulted in a TPC of 2.95 ±0.03 g glucose kg −1 VS , with no significant differences between results. Similar results were recorded for the FAR aqueous extracts, however, the differences between the ethanolic extracts were significantly higher. The ethanolic extraction of FAR after a first aqueous extraction (FAR-WE) process yielded 56 % more TPC than the single ethanolic extraction (FAR-E). This could be explained by the degree of polymerization of the phenolic compounds and the strength of the bonds with other molecules. For example, phlorotannins, as the most common phenolic compound in brown marine algae, are usually present in a cell vacuole, named physode and are accumulated in the outer cortical layer of the thalli (Imbs and Zvyagintseva, 2018). Moreover, these compounds tend to form a complex with alginic acid, which is a common carbohydrate responsible for the structure of the cell wall (Imbs and Zvyagintseva, 2018). Thus, it could be argued that a first aqueous extraction weakens this complex, allowing for a higher TPC yield after the use of ethanol. Moreover, as discussed above, the natural degradation of the algae helps reducing the carbohydrate content and increasing the breakdown of these compounds, allowing for a greater phenolic release in comparison with the alive fresh algae in its natural environment, which could compensate for the degradation of the phenolic compounds during the natural degradation process. This could explain the lack of significant differences in the TPC of both FR and FAR samples when extracted firstly with water and subsequently with ethanol. Regarding the RS yields, from Table 3 it could be observed that the sequential extraction process of ethanolic and further aqueous extraction methodology enhance the RS recovery greatly. In fact, the FR-EW extract yielded 2.4 times more RS than the FR-W extract. Mono and oligo saccharides are relatively soluble in water, which suggests that the carbohydrate fraction that remains in the solid residue is composed of higher-complex carbohydrates, such as cellulose or hemicellulose, bonded with other non-soluble compounds, such as phlorotannins (Imbs and Zvyagintseva, 2018). The preliminary ethanolic extraction process could further damages the cell wall allowing for an easier incursion of the water through the cell. In fact, a recent study on the effect of ethanol in E. coli showed that ethanol reduced the cell membrane integrity; increased the stress, through the higher production of reactive oxygen species, causing subsequent damage to the DNA; and, generally, disrupted the normal cell behaviour (Cao et al., 2017). Additionally, and similar to the single extraction process, the RS content obtained from the FR samples after aqueous extraction was higher than that obtained from the FAR samples, due, most likely to the natural degradation process that takes place when the alga is settled and accumulated on the beach. 3.1.5. Antioxidant capacity of the Rugulopteryx okamurae extracts The antioxidant capacity of the extracts was assessed by performing the FRAP and the DPPH RSA tests. Results are shown in Table 3. Surprisingly, FRAP results from the aqueous extract were higher than those from the equivalent ethanolic extracts. For example, while the aqueous extract from the FAR biomass reported a FRAP value of 1.50 ±0.04 mM Trolox eq., the ethanolic extract only gave 0.76 ±0.05 mM Trolox eq. Although this behaviour was observed regardless of the biomass (i.e. FR or FAR), the differences between the aqueous and the ethanolic extract were smaller in the FR samples. Moreover, while both aqueous extract (FR-W and FR-EW) showed lower FRAP values than their FAR equivalents, the opposite was observed with the ethanolic extracts (FR-E and FR-WE). If the TPC and the RS contents are taken into consideration it could be concluded that all these results suggested that the antioxidant capacity is primary given by a phenolic compound or compounds that shows higher affinity to water than to ethanol or by small oligosaccharides detected by the RS test. Finally, similar to what was observed from the TPC and RS extract yields, both aqueous and ethanolic extracts of the second extraction process showed higher values than their equivalents single extracts. This supports the idea of cell disruption after a first extraction process. This is the first time that the antioxidant capacity of R.o. aqueous and ethanolic extracts is reported by using the FRAP test. Results obtained in this study (0.76 ±0.05–1.56 ±0.01 mM Trolox eq.) were in the upper range of other marine seaweed. For instance, Tolpeznikaite et al. (2022) assessed the FRAP of the ethanol/water (70:30 v/v) extract of the algae Cladophora rupestris, Cladophora glomerata, Ulva intestinalis and Furcellaria lumbricalis reporting values of 0.077, 0.360, 0.869 and 0.063 mM Trolox eq., respectively. Ummat et al. (2020) also reported the FRAP value of 11 brown seaweeds extracted with ethanol/water (50:50). However, this study reported the FRAP values as mg trolox eq. per gram D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 7 of dry weight extract (dwe), which do not allow for the comparison with the previously mentioned article. Nevertheless, the reported values (from 5 to 65 mg Trolox eq. g −1 dwe ) are lower to those observed in the present study for the alga R.o. (from 97 to 689 mg Trolox eq. g −1 dwe ). The authors are aware that there are more articles reporting the FRAP value of ethanolic and aqueous extract of marine seaweeds, however, the authors would like to highlight the inconsistencies on the FRAP methodologies reported and the lack of standardized units which preclude the comparison between studies when not enough data are provided. In any case, the aqueous extract of the FAR biomass seems to present one of the highest FRAP value reported in the literature for marine seaweeds. Regarding the DPPH radical scavenging activity of the studied extracts (Table 3), the tendency was similar to that observed for the FRAP test. DPPH RSA values were lower in the aqueous extracts than in their ethanolic equivalents, which means that less dried extract is needed to reduce the DPPH absorbance to half its value. These results confirmed that the compounds with the highest antioxidant capacity from the alga R.o. presented higher affinity to water than to ethanol. Moreover, with only one exception (i.e. FR-EW), the higher the RS content was, the lower the DPPH RSA . While, by contrast, the higher the DPPH RSA was, the higher the TPC yield. This, as observed from the FRAP test, strongly suggested that these antioxidant compounds are phenols or other compounds that shows higher affinity to water than to ethanol or small oligosaccharides detected by the RS test. Similar to the FRAP, the DPPH RSA results from the ethanolic extracts were not significantly different regardless the biomass was extracted once or twice. However, the DPPH RSA was higher in the FAR-W extract than in the two-step extract FAR-EW, while the opposite was observed for the other two aqueous extracts FR-W and FR-EW. Moreover, the DPPH RSA of the FR extracts were higher than the equivalent extracts from FAR samples, with the exception of the one-step aqueous extracts which showed no significant differences. Similar to the FRAP test, different studies reported DPPH RSA values in units which are not easily to use for comparison, due to, mainly, the methodology followed or the expressed units. For example, Vega et al. (2022), reported values of DPPH for different extracts of R.o. in the range 5–25 μ mol Trolox g −1 dwe . However, L´ opez-Hortas et al. (2023) reported the DPPH value as the inhibition percentage (IP) of the ethanolic (50 %) extract of R.o., which varied along with the biomass drying system (Oven: 4.6 %; MHG: 6.1 %). Table 3 shows the DPPH RSA values as the minimum dried extract required to reduce the DPPH absorbance by half (IC 50 ), which could be interpreted as an IP of 50 %. However, this IP is dependent on the DPPH solution concentration, the solvent and the extract-to-DPPH ratio used during the absorbance measurement. Thus, a direct comparison between the L´ opez-Hortas et al. (2023) values and those reported in this study would be not possible. Nevertheless, a rough comparison could be made if the results are expressed per the amount of dried extract needed to reduce a certain amount of DPPH. L´ opez-Hortas et al. (2023) reported TS concentration of 0.479 and 1.316 mg TS ml −1 extract for the biomass dried by oven and MHG, respectively. Moreover, the DPPH assay was performed by measuring the absorbance drop when 50 μ l of the extract was added to 2 ml of 0.06 mM DPPH solution. This means that, in the case of oven dried R.o., 23.96 ng of dried extract reacted with 2.176 ng of DPPH, and in the case of MHG, 65.8 ng of dried extract reacted with 2.886 ng of DPPH. If we normalize this to the amount needed to react with 1 mg of DPPH, then 11.010 and 22.800 mg of dried extract is requested for the dried biomass by oven or MHG, respectively. In our study, this relation ranged from 12.75 to 37.72 mg of dried extract, being the lower value obtained from the aqueous extract of the residue previously extracted with ethanol (FAR-EW). Other macroalgae also show good antioxidant activity, although, in most of the cases, with DPPH RSA values higher than those reported for the R.o. For example, the DPPH RSA IC 50 of the green algae Caulerpa racemose and Caulerpa sertularioides were 456.1 and 180.9 μ g ml −1 , respectively (Sanniyasi et al., 2023). Goutzourelas et al. (2023) also studied the antioxidant activity of 14 marine macroalgae methanolic extracts, reporting that the lowest value observed was 0.31 mg ml −1 for the species Gigartina teedei. On the other side, the brown macroalgae Padina boergesenii showed values of 36.75 and 42.784 μ g ml −1 for its ethanolic and methanolic extracts, respectively (Kalasariya et al., 2023), and similar results were reported for Padina australis (16.63 μ g ml −1 ) and Turbinaria ornata (43.22 μ g ml −1 ) (Warouw et al., 2022). 3.2. Biochemical methane potential test The main physicochemical characteristics of the substrates used for the BMP tests are described in Table 1. Furthermore, the elemental analysis of the non-extracted biomasses is displayed in Table S1, while for the extracted solid residue is shown in Table S3 of the supplementary material. Previously reported studies where the biochemical methane potential of the algae Rugulopteryx okamurae were also tested showed a VS/TS ratio around 60–70 % (De la Lama-Calvente et al., 2023a; De la Lama-Calvente et al., 2023b; De la Lama-Calvente et al., 2021). However, the batches tested in this study showed a significant higher ratio (87–91.5 %). Subsequently, the mineral fraction of the biomasses used in the present study were significantly lower than those previously reported. Part of this mineral reduction could be related to the washing of the biomasses with tap water (Table S1), which were not done in the previous studies, and a further depletion due to the extraction process (Table S3). However, these processes cannot be accounted as the sole reason. When comparing the results of the un-washed biomasses with those reported in De la Lama-Calvente et al. (2023b), some of the macro elements already showed significant differences. For example, the Ca content of the natural R.o. varied from 19.6 ±0.7 in the present study to 89 ±3 in De la Lama-Calvente et al. (2023b). This confirms that the mineral content of natural biomasses varies greatly depending on the several factors, such a seasonality, climatology, etc. It was also observed, that the contents of the potentially toxic elements were significantly lower than those reported previously for the macroalgae R.o. (De la Lama-Calvente et al., 2023a; De la Lama-Calvente et al., 2023b). In fact, some elements (Pb, Cu and Cd) that have been directly linked to the inhibition of previously reported assays, have been found to be below the reported toxic limit in this study. For example, while the toxic limit for Pb have been stablished at 1.27 ppm (Alrawashdeh et al., 2020), the content found in the liquid fraction of the reactors was in the range of 17–65 ppb. However, some other elements (Fe, Ni, Cr, Hg) were found to be higher than the stablished toxic limit for AD. For example, the soluble Ni content in the reactor was in the range of 57–330 ppb, while the toxic limit reported by Alrawashdeh et al. (2020) was stablished at 41 ppb. This suggest that although the AD could be potentially inhibited by these elements, the methane yield would not be as affected as previously reported (De la Lama-Calvente et al., 2023b). In fact, based on the main physicochemical characteristics of the digestates after the BMP test (Table 2) no major disruption of the anaerobic performance was observed. pH values (7.64–7.90) were within the optimum range for AD performance (Holliger et al., 2016). TA values (3550–4250 mg CaCO 3 kg −1 ) were below the limit considered for buffer unbalance (Holliger et al., 2016). Similarly, TAN (590–760 mg N-NH 3 kg −1 ) were below the established limit (2000 mg N-NH 3 L −1 ) to consider a process inhibition due to the presence of free ammonia (Yenigün and Demirel, 2013). The organic fraction, measured as either VS or CODt, showed certain differences between the tests which could be directly linked to the produced methane, indicating that the higher the methane yield the lower the organic fraction remaining in the final digestate. Although no great significant differences were observed from the physicochemical parameters of the digestates, the methane yield showed a different tendency (Table 2 and Fig. 2). Regardless of the biomass origin (i.e.: FR or FAR), as the alga was further extracted the methane yield increased. For example, the extraction of bioactive compounds of FR using water provoked a methane increase of 28 %, D. De la Lama-Calvente et al. Journal of Environmental Management 370 (2024) 122504 8 Fig. 2. Accumulated methane of the untreated and extracted algae biomasses versus time. Bars represent standard deviation. Fresh ashore Rugulopteryx okamurae (FAR) extracted with water (FAR-W) or ethanol (FAR-E) or with water and then with ethanol (FAR-WE) or firstly with ethanol and then with water (FAR-EW). Fresh Rugulopteryx okamurae (FR) extracted with water (FR-W) or ethanol (FR-E) or with water and then with ethanol (FR-WE) or firstly with ethanol and then with water (FR-EW). Table 4 Methane yield of the algal residue after added-value compounds recovery. Added-value compound AD System Methane Yield of residue (L CH4 kg −1 VS added ) Methane Efficiency Source Green Macroalgae Ulva lactuca Sap BMP 206 98 % Mhatre et al. (2019) Ulvan BMP 226 107 % Mhatre et al. (2019) Protein BMP 229 109 % Mhatre et al. (2019) Sap and ulvan BMP 409 195 % Mhatre et al. (2019) Sap, ulvan and protein BMP 323 154 % Mhatre et al. (2019) U. rigida Alginic acid, fucoidan, fucoxanthin, laminarin, mannitol, proteins BMP 73 –Tedesco and Stokes (2017) Red Macroalgae Gracilaria manilaensis Agar BMP 191 85 % Hessami et al. (2019) G. multipartita Agar BMP 253 131 % Abomohra and Almutairi (2020) Abomohra and Almutairi (2020) Agar and Acid pretreatment BMP 279 144 % G. persica Agar BMP 148 83 % Hessami et al. (2019) Brown Macroalgae Ascophyllum nosodum Alginic acid, fucoidan, fucoxanthin, laminarin, mannitol, proteins BMP 84 –Tedesco and Stokes (2017) Ecklonia maxima Polysaccharide BMP 862 –Darko et al. (2022) Fucus serratus Alginic acid, fucoidan, fucoxanthin, laminarin, mannitol, proteins BMP 101 –Tedesco and Stokes (2017) F. vesiculosus Alginic acid, fucoidan, fucoxanthin, laminarin, mannitol, proteins BMP 103 –Tedesco and Stokes (2017) Laminaria digitata Succinic Acid BMP 285 100% Alvarado-Morales et al. 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